CAPITULO I. LA MINERÍA INFORMAL - ILEGAL EN LA CUENCA DEL RÍO
I.1. d. ORGANIZACIÓN DE LOS MINEROS - LAS COOPERATIVAS
Since carvacrol resulted the most effective compound against Salmonella spp. and Listeria monocytogenes from agar disk diffusion test, films prepared with two different concentrations, 7 and 10%, were applied to real food matrices, chicken breast and salmon fillets. Antimicrobial activity resulted different as a function of the carvacrol concentration.
The tests performed on chicken and salmon packaged with WG film with carvacrol 7% did not show an antimicrobial effect against both pathogenic microorganisms considered (data not shown).
Concerning the films prepared with 10% of carvacrol, in the first batch of chicken breast packaged in direct contact and with headspace, Salmonella spp. count in the untreated samples remained more or less constant (P>0,05) during the first three days of shelf life and then increased on the fifth day. In headspace treated samples, the pathogenic load decreased during the storage time, reaching the lowest value after five days (Figure 3A); in the case of direct contact samples, Salmonella spp. counts kept unchanged during storage time with a lower concentration compared to control sample.
In the second batch, the amount of Salmonella spp. in treated samples was lower than in control samples at each analytical session and for both ways of packaging.
Just as for the first batch, the maximum inhibitory effect of carvacrol film was observed on the fifth day (Figure 3B).
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Figure 3. Enumeration of Salmonella spp. observed in chicken breast packaged with WG film (untreated) and with 10% carvacrol WG film (treated), batch 1 (3A) and batch 2 (3B). Means values obtained from three replicates.
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As regard smoked salmon artificially contaminated with Listeria
monocytogenes, two batches of samples were considered. In batch 1 (Figure
4A), a significant increase in pathogen counts in untreated samples was observed on the third day of shelf life, compared to the samples packed with films containing carvacrol, both in head space packaging and in direct contact with food. After three days of storage a decrease in microbial counts was observed also in the untreated sample. The antimicrobial effect was confirmed in batch 2 (Figure 4B), where an increase in microbial counts in untreated samples compared to treated samples was observed after three and five days of storage, for both headspace and direct contact samples. Results obtained show different behavior for treated and untreated samples: in fact, when for untreated samples Listeria monocytogenes counts increased as a function of storage time, for treated samples values are settled on a constant value. This evolution for treated samples instead of a decrease in microbial counts suggested an inhibitory effect of the aroma compounds: therefore, we could hypothesize that carvacrol cannot cause a diminution of microorganisms (i.e. sterilizing effect) but its presence in the headspace probably create a disadvantageous environment for microbial growth.
No differences between the two kinds of packaging both for controls and for treated samples were observed in the second batch. The load of Listeria
monocytogenes observed in salmon packaged with WG film, increased
significantly (P<0,05) after the third day of shelf life and maintained a constant value until the fifth day, while the pathogen counts in treated samples were constant during the whole observation period. The reduction of microbial loads induced by carvacrol packaging were more evident than in batch 1.
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Figure 4. Enumeration of Listeria monocytogenes observed in smoked salmon packaged with WG film (untreated) and with 10% carvacrol WG film (treated), batch 1 (4A) and batch 2 (4B). Means values obtained from three replicates.
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Two different packaging methods were compared in Table 5 and 6. Results showed a difference in microbial inhibition as a function of the WG film position: in particular, a greater microbial inhibition was found when the specimen was set on the lid, not in contact with the sample. Therefore, carvacrol seemed to have an effect against Salmonella spp. when it diffused in the headspace.
Table 5. Antimicrobial efficacy against Salmonella spp. in chicken breast as a function of 10% carvacrol WG film position in the package.
Food products
Shelf life (days)
WG film with carvacrol 10% (headspace)
WG film with carvacrol 10% (direct contact) Chicken breast (batch 1) 1 5,18a 5,05a 3 5,01a 4,91b 5 4,90a 5,09b Chicken breast (batch 2) 1 5,53a 5,55a 3 5,44b 5,50a 5 5,03a 5,05a
Table 6. Antimicrobial efficacy against Listeria monocytogenes in smoked salmon as a function of 10% carvacrol WG film position in the package. Food
products
Shelf life (days)
WG film with carvacrol 10% (headspace)
WG film with carvacrol 10% (direct contact) Smoked salmon (batch 1) 1 5,47a 5,49a 3 5,47a 4,48a 5 4,39a 5,08b Smoked salmon (batch 2) 1 5,54a 5,55a 3 5,51a 5,57a 5 5,52b 5,56a 3.4 Mechanical properties
Mechanical properties of an edible film are very important because they determine the properties of elasticity, strength and plasticity in order to maintain its integrity during handling and storage. In order to understand if the addition of essential oils or their components can affect the mechanical properties of the film as a function of the type of essential oil, mechanical properties of WG prepared with the same content of carvacrol, eugenol, trans-cinnamaldehyde were measured and compared with a sample prepared without EO addition (WG). Results of tensile strength and elongation are reported in Figure 5, A and B. Tensile strength (Figure 5A) was not affected by the type of aroma compounds, unlike what was for elongation (Figure
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5B): WG containing eugenol was characterized by the highest elongation, where the sample prepared with the addition of cinnamaldehyde shown the lowest elongation. In the first case, results suggest a plasticizing effect of eugenol, contrary to cinnamaldehyde that seemed to reinforce the gluten network acting as a cross-linker, as previously observed by Balaguer et al. (2011).
Figure 5. Tensile strength (A) and elongation (B) of WG films with and without aroma compounds.
0 1 2 3 4 5 6 7 tens il e streng ht (M P a ) aroma compounds 3%
wg eugenol carvacrol cinnamaldehyde
0 20 40 60 80 100 120 140 160 180 200 el o ng a ti o n (% ) aroma compounds 3%
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Mechanical properties of WG prepared with different concentration of added carvacrol (3, 5, 7, 10%) are measured and compared. Results of tensile strength (Figure 6) showed no effect on film mechanical properties with EO initial concentration up to 7%. With EO concentration higher than 7%, it seemed that carvacrol had a cross-linker effect on the protein.
Figure 6. Tensile strenght of WG films at different concentration of carvacrol.
Elongation results (Figure 7) confirmed the data obtained for tensile strength, suggesting a reduction in plasticity and an increased rigidity of the structure.
Figure 7. Elongation of WG films at different concentration of carvacrol.
b b a b 0 0,5 1 1,5 2 2,5 3 Tens il e streng ht (M P a ) carvacrol Tensile strenght WG with carvacrol
3% 5% 7% 10% a a b b 0 50 100 150 200 250 El o n ga ti o n % carvacrol Elongation WG with carvacrol
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4 DISCUSSION AND CONCLUSIONS
The preliminary study for the in vitro evaluation of the antibacterial activity of WG film with aroma compounds at different concentrations showed a positive effect only for WG film with carvacrol 7% and 10%. No effect was observed for gluten based edible films with cinnamaldehyde or eugenol, regardless the essential oil concentration incorporated. Films prepared with the lowest carvacrol concentrations (3-5%) did not show antimicrobial effect. Our results did not agree the data obtained by Fernández-Pan et al. (2012), that demonstrated effectiveness WPI films containing oregano against Listeria innocua, Salmonella Enteritidis and Staphylococcus aureus when the active substance concentration was 1%. Our results did not confirmed what found by Hosseini et al. (2008), in which edible films containing 0,5% clove essential oil were effectives against Listeria
monocytogenes and Staphylococcus aureus but they resulted in non-
effectiveness against Salmonella Enteritidis and Pseudomonas aeruginosa. As concern the challenge tests with real matrices packaged with carvacrol film, a significant inhibitory effect on the growth of the pathogen was observed only if carvacrol concentration was at least 10%. The tests performed on chicken and salmon packaged with WG film with carvacrol 7% showed a not significant antimicrobial effect against both pathogenic microorganisms considered.
For chicken breast the maximum effect was obtained after five days both for packaging with head space and direct contact. In the first batch carvacrol incorporated in head space packaging seemed to be more efficient than if placed in direct contact with food matrix, but it was not confirmed in the second batch where the action was similar.
In smoked salmon the effect of reduction on Listeria monocytogenes loads exerted by experimental carvacrol film was more evident in the second batch and it seemed to be independent in the way of food packaging, i.e. with head space or direct contact.
The results from this work are coherent with previously published research (Ravishankar et al., 2009) although the inhibition of pathogen growth is not so marked. In Ravishankar’s study, carvacrol incorporated into edible apple film showed antimicrobial activity against Salmonella Enterica on the surface of chicken breast and Listeria monocytogenes on the surface of ham. At 4°C, films contain 0,5% carvacrol inducing about 0,8 log reduction of Salmonella Enterica. At 4 °C, film containing carvacrol showed about 2, 1, and 0,5 log reductions in Listeria monocytogenes populations and at 3%, 1,5%, and 0,5% concentrations, respectively.
The different results found by other researchers can be explained by the composition of the film matrices, which has also a fundamental effect on their antimicrobial activity. Emiroglu et al. (2010) observed antimicrobial
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activity against Staphylococcus aureus, Escherichia coli, Escherichia coli O157:H7, Pseudomonas aeruginosa and Lactobacillus plantarum with 1% oregano essential oil encapsulated in soy protein isolated based edible films, while Pellissari et al. (2009) inhibited the Salmonella Enteritidis and
Staphylococcus aureus strains effectively by using a starch-chitosan matrix
with a lower essential oil content (0,5%).
When a packaging material is developed, different properties have to be considered for final application. Among these, mechanical properties can be crucial, especially in the case of a bilayer or multilayer packaging, where the compatibility between the different materials is affected also by elongation and tensile strength, in particular when multilayer packaging is composed by a bio-based layer. Different papers discussed the effect of aroma compound addition on the bio-based film mechanical properties: in particular, the addition of trans-cinnamaldehyde to gluten-based films modified mechanical properties of final film, suggesting a role of the aroma in reinforcing the network formed by the protein (Balaguer et al., 2011a). Along with the study of antimicrobial activity, mechanical properties of films containing aroma compounds were investigated, in view of the application to develop a real packaging system.
The addition of aroma compounds seemed to affect gluten films mechanical properties differently as a function of the type of the aroma compound: in particular the elongation resulted the highest when eugenol was added to the film. Since carvacrol resulted as the most effective against Salmonella spp. and Listeria monocytogenes growth, the effect of different concentrations of this aroma compounds on mechanical properties was deepened. Even if both tensile strength and elongation changed as a function of aroma compounds concentration, films prepared with 10% of initial content of aroma compound seemed to show mechanical properties not significantly different from those of film prepared with 3% and 5% of initial aroma compound content. Therefore, mechanical properties of film with increased antimicrobial activity were not affected by aroma compounds concentration.
Due to the microbiological results, we focused this study on the carvacrol and we observed that the addition of carvacrol in WG can modulate the structural characteristics of the mechanical properties of these films.
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5 REFERENCES
AFNOR BRD 07/06-07/04 - PCR iQ-CHECK SALMONELLA II kit (BIO-RAD)
AFNOR BRD 07/10-04/05 - PCR iQ-CHECK LISTERIA MONOCYTOGENES II kit (BIO-RAD)
Balaguer M.P., Gomez-Estaca J., Gavara R., Hernandez-Munoz P., 2011a. Biochemical properties of bioplastics made from wheat gliadins cross-linked with Cinnamaldehyde. Journal of Agricultural
and Food chemistry, 59, 13212-13216.
Ben Arfa A., Combes S., Preziosi-Belloy L., Gontard N., Chalier P., 2006. Antimicrobial activity of carvacrol related to its chemical structure. Letters in Applied Microbiology, 149-154.
Burt S., 2004. Essential oils: their antibacterial properties and potential applications in food – a review. International Journal of
Food Microbiology. 94:223-253.
CLSI, 2012. Methods for dilution antimicrobial susceptibility testes for bacteria that grow aerobically; approved standard – ninth edition. Clinical and Laboratory Standard Institute, M07-A9, 32(2), replaces M07-A8, 29(2).
Emiroglu Z.K., Yemis G.P., Coskun B.K., Candogan K., 2010. Antimicrobial activity of soy edible films incorporated with thyme and oregano essential oils on fresh ground beef patties. Meat Science. 86:283-288.
EURL Lm Technical Guidance Document for conducting shelf-life studies on Listeria monocytogenes in ready-to-eat foods, 2014. Version 3, 1-47.
European Food Safety Authority (EFSA) 2015. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-born in 2013. EFSA Journal. 13(1):3991 (under review). Fernández-Pan I, Royo M., Maté J.I., 2012. Antimicrobial activity of
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spoiler and foodborne pathogens. Journal of Food Science. 77(7):383- 390.
Gutiérrez L., Batlle R., Sánchez C., Nerín C., 2010. New approach to study the mechanism of antimicrobial protection of an active packaging. Foodborne Pathogens and Disease. 7(9):1063-1069. Hosseini M.H., Razavi S.H., Mousavi S.M.A., Yasaghi S.A.S.,
Hasansarei A.G., 2008. Improving antibacterial activity of edible films based on chitosan by incorporating thyme and clove essential oils and EDTA. Journal of Applied Science. 8:2895-2900.
ISO 11290-2:1998 – Microbiology of food and animal feeding stuffs – Horizontal method for the detection and enumeration of Listeria monocytogenes. Part 2: Enumeration medium.
Iturriaga L., Olabarrieta I., De Marañón I. M., 2012. Antimicrobial assays of natural extracts and their inhibitory effect against Listeria
innocua and fish spoilabe bacteria, after incorporation into
biopolymer edible film. International Journal of Food Microbiology. 158:58-64.
Marcuzzo E., Peressini D., Debeaufort F., Sensidoni A., 2010. Effect of ultrasound treatment on propertiesof gluten-based film. Innovative
Food Science and Emerging Technologies. 11:451-457.
Munmaya M., 2015. Handbook of encapsulation and controlled release. CRC Press Taylor & Francis Group.
Pelissari F.M., Grossmann M.V.E., Yamashita F., Pined E.A.G., 2009. Antimicrobial, mechanical, and barrier properties of cassava starch-chitosan film incorporated with oregano essential oil. Journal
of Agricultural and Food Chemistry. 57:7499-7504.
Ravishankar S., Zhu L., Olsen C.W., McHugh T.H., Freidman M., 2009. Edible apple film wraps containing plant antimicrobials inactivate foodborne pathogens on meat and poultry products. Food
Microbiology and Safety. 74(8):440-445.
Sanla-Ead N., Jangchud A., Chonhenchob V., Suppakul P., 2011. Antimicrobial activity of cinnamaldheyde and eugenol and their
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activity after incorporation into cellulose-based packaging films.
Packaging Technology and Science. 25:7-17.
Seydim A.C., Sarikus G., 2006. Antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and garlic essential oils. Food Research International. 39:639-644.
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CONCLUSIONS
Antimicrobial packaging is a promising and rapidly emerging technology in which antimicrobial agents are incorporated into or coated onto food packaging materials in order to prolong shelf life of the packaged food or to control the growth of pathogenic microorganisms. Even when antimicrobial films fail to remove completely higher numbers of unwanted microbes, they can act as an additional and post processing safety measure.
The first part of study aimed to verify the antimicrobial effectiveness of a commercial antimicrobial packaging (Food-Touch®) against the spoilage microorganisms of a fresh cheese. The microbial, chemical and sensorial characteristics of active packed Stracchino cheese were compared with ones observed in the cheese wrapped with a traditional passive packaging and in cheese packaged with an innovative film (Ovtene®), at that time regularly used in the production plant. The cheese manufacturer had previously replaced the traditional passive packaging with Ovtene® because the innovative film kept longer the cheese freshness and preserved its organoleptic characteristics, prolonging the shelf life. All the Stracchino samples, with active or not active packaging, belonged to the same batch of production and were hand-packaged by the producer as usually; so no production differences affected the results apart from the kind of film used as packaging.
The packaging products were analysed by mimicking a real-life scenario, in terms of time and temperature of storage as established by manufacturer, and also because a real food – Stracchino cheese – was used. Despite the Food-
Touch® system resulting in lower bacterial growth at some given times
throughout the cheese storage, the final results did not show any significant difference in the cheese microbiota examined, of any packaged Stracchino cheese samples, excluding that Food-Touch® and Ovtene® systems exerted a different inhibitory effect on the growth of spoilage microorganisms.
On the contrary, a putative effect exerted by the Ovtene® system, which maintained two of the examined sensory characteristics, homogeneity and adherence, was observed. This effect may have been a consequence of the preservation of the functional cheese microbiota, known to be involved in the typicalorganoleptic properties of cheeses.
These results are coherent with previously published research, suggesting that although application of silver based antimicrobial systems in the food industry is a widespread phenomenon, appraisal of the full potential of silver as an antimicrobial and its possible implementation in food packaging technologies is still a challenging task.
Our results pose some safety concerns, as the level of silver ions migration from the active packaging system containing silver exceeded the maximum
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established level for the migration of a non-authorised substance through a functional barrier (Commission regulation (EC) No. 450/2009).
Despite the relevance of the topic, to date, only a limited number of reports have studied the potential for silver migrating from plastic food containers, with most reports being focused on silver nanoparticles.
In the second part of the thesis the antimicrobial activity of an experimental gluten-based edible film added with carvacrol, known for its antimicrobial capacity, was tested against two foodborne pathogens, Salmonella spp. and
Listeria monocytogenes on fresh poultry and smoked salmon respectively.
Even if several studies about the efficacy of essential oils or their components against microbial spoilage were previously conducted, very few focused on the activity of these molecules once incorporated in a packaging material and applied to real food products.
As concerns the outcome of the study, a significant inhibitory effect on the growth of the pathogens was observed only if carvacrol concentration was at least 10%. The tests performed on chicken and salmon packaged with WG film with carvacrol 7% showed a not significant antimicrobial effect against both pathogenic microorganisms considered.
The results from this work are coherent with previously published research although the inhibition of pathogen growth is not so marked. The different results found by other studies can be explained by the composition of the film matrices, which has also a fundamental effect on their antimicrobial activity. The final content of aroma compound incorporated in the film should be determined in order to investigate the real conditions under which the aromas are effective against the microorganisms selected. In fact the quantity of aroma compounds added to the packaging material should not be too pronounced to avoid a significant and unwanted change in the original sensory characteristics of the product.
The effect of the aroma compound incorporated in the packaging film seems to be linked to the ability of creating an unfavourable environment for the microorganism multiplication, saturating the internal packaging atmosphere, rather than a bactericidal action. So to ensure such effect throughout the food product shelf life it would be necessary to achieve a controlled release of the active molecule in order to have a constant concentration over time and to avoid the aroma consumption in the first phase of the storage.
In conclusion, in this study the efficacy of active packaging systems, both packaging material with silver zeolites and with aroma compounds, was evaluated in real food products stored at refrigeration temperature, 0-4°C. This temperature was selected in order to simulate the effective storage conditions of the products studied (Stracchino cheese, chicken and salmon fillets).
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This represents an innovative approach both because we considered real food matrices and because the refrigeration temperatures are different compared to those used in vitro tests.
Moreover the volatility of the aromatic compounds is conditioned by the temperature, so the effectiveness of the experimental packaging systems is even more interesting because evaluated in unfavourable operating conditions.
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